Peptides are used by cells from yeast to mammals to elicit physiological responses. The use of peptides as messengers usually involves the following steps: 1) production and release of the peptide by a specific cell, 2) interaction of the peptide with a receptor on the surface of the target cell, and 3) degradation of the peptide to terminate its action. The first and last steps of this scheme require the participation of proteases/peptidases. There is increasing evidence that membrane-associated zinc-metallopeptidases play important roles in both of these steps. Although activation of prohormone precursors into bioactive peptides is generally performed by proteases of the subtilisin family located in the Trans-Golgi Network or in secretory granules of the cell (for a review see: (Seidah and Chretien, 1995)) a few peptides need a final processing step. This step involves the action of membrane-associated zinc-metallopeptidases. Two cases are particularly well documented: angiotensin-converting enzyme (ACE) which cleaves inactive angiotensin I into angiotensin II (Corvol and Williams, 1997) and endothelin-converting enzymes (ECEs) which cleave isoforms of big endothelins into endothelins (Turner, 1997a). In addition to their role in peptide activation, cell surface zinc-metallopeptidases have also been implicated in the termination of the peptidergic signal by breaking down the active peptides into inactive fragments. One of the best known of these peptidases is probably Neutral Endopeptidase-24.11 (Neprilysin, NEP) that has been implicated in the physiological degradation of several bioactive peptides (Kenny, 1993). Interestingly, NEP and the ECEs show significant structural similarities and appear to be members of a family of peptidases that also includes PEX, a newly discovered and not yet characterized peptidase, and the KELL blood group protein (Turner and Tanzawa, 1997b). Because of their important role as regulators of bioactive peptide activity, these enzymes (more specifically NEP and the ECEs) have been identified as putative targets for therapeutic intervention, similar to the way ACE inhibitors are used to control blood pressure. The recent discovery of PEX, another member of the family, which appears to be involved in phosphate homeostasis, raised the possibility that other yet unknown members might exist.
Members of the NEP-like family are type II membrane proteins consisting of three distinct domains: a short NH2-terminal cytosolic sequence, a single transmembrane region, and a large extracellular or ectodomain responsible for the catalytic activity of the enzyme. There are potential N-glycosylation sites and cysteine residues that are involved in disulfide bridges stabilizing the conformation of the active enzyme. These enzymes are metalloenzymes with a Zn atom in their active site. As such, they belong to the zincin family of peptidases which is characterized by the active site consensus sequence HEXXH (Hooper, 1994), where the two histidine residues are zinc ligands. In members of the NEP-like family of peptidases, the third zinc ligand is a glutamic acid residue located on the carboxy-terminus side of the consensus sequence. This characteristic puts them in the gluzincin sub-family (Hooper, 1994). The model enzyme for gluzincins is thermolysin (TLN) a bacterial protease whose 3D structure has been determined by X-ray crystallography (Holmes and Matthews, 1982). The active site of NEP has been extensively studied by site-directed mutagenesis and several residues involved in zinc binding (Devault et al., 1988b; Le Moual et al., 1991; Le Moual et al., 1994), catalysis (Devault et al., 1988a; Dion et al., 1993), or substrate binding (Vijayaraghavan et al., 1990; Beaumont et al., 1991; Dion et al., 1995; Marie-Claire et al., 1997) have been identified (for a recent review see Crine et al., 1997).